While studying whether compounds known to affect PPARgamma could play a role in inflammatory bowel diseases, a team at the University of Rochester Medical Center found that medium-to-high doses of PPARgamma inhibitor killed colorectal cancer cell lines. Despite the compound's class name, the anti-cancer effect has nothing to do with the ability of the compounds to inhibit PPARgamma function. Researchers believe that PPARgamma inhibitors instead attack the "skeletons" of cancer cells that enable them to reproduce, grow and spread. Better solutions are needed because, according to the American Cancer Society, colorectal cancer remains the no. 2 cause of cancer death for men, and the no. 3 cause of cancer death for women.
"This is the first observation of a small molecule dramatically reducing levels of the proteins called tubulins, the building blocks of cancer cell skeletons," said Katherine L. Schaefer, Ph.D., a research assistant professor within the Department of Medicine, Gastroenterology and Hepatology Division, at the University of Rochester Medical Center, and first author of the paper. "Because cells that line the colon are similar to those in the liver, esophagus and skin, we see potential for a new way to treat those cancers as well."
In the study that led to the discovery of the anti-cancer effect, researchers were looking for new ways to reduce inflammation seen in Crohn' s disease and ulcerative colitis, bowel diseases that cause pain and diarrhea. Specifically, they were comparing the effect on inflammation of encouraging the action of the PPARgamma protein (with activator compounds) against discouraging it (with inhibitors). The team conducted these experiments using colorectal cancer cells as study models because they arise from normal gut cells and share some of their qualities (e.g. normal inflammatory signals). Unlike normal gut cells, however, cancer cells do not die when removed from the gut wall. Living on in the absence of normal survival signals makes cancer cells dangerous in the body, but useful as cell lines for study.
While comparing PPARgamma activators and inhibitors, Schaefer noted with frustration that her cancer cells were dying before she could complete her experiments. Retracing her steps, she found that she had used too much inhibitor. The team, led by Lawrence J. Saubermann, M.D., associate professor of Medicine at the Medical Center, realized they had come across a potentially new therapeutic effect, and launched experiments to confirm it.
In the newly published study, researchers observed the effects of three compounds known from the literature to inhibit PPARgamma, T0070907, GW9662 and BADGE, on the ability of colorectal tumor cells to survive. High doses (10-100 ìM) of all three interfered with colorectal cancer cell growth, reduced the cells' ability to spread through the bloodstream to cause new tumors elsewhere (metastasize) and caused cells to self-destruct, generally within 24 hours. Further experiments showed that high-dose PPARgamma inhibition destroyed cancer cell microtubules, protein structures that form part of the skeleton of cells.
Beyond providing structural support and shape to cells, microtubules expand and shrink to generate the force needed for cells to divide, a basic process in tumor growth. Microtubules are made of two rel ated proteins, alpha and beta tubulin, which pair up to form a chain and then wind into a helix. The current studies found that high-dose PPARgamma inhibitors reduced levels of alpha and beta tubulin by 60 to 70 percent. Also described in the publication are studies where PPARgamma inhibitors killed tumor cells in mice without causing significant toxicity. That provides at least the hope that the drug class may not be prohibitively toxic in humans, the researchers said. More formal toxicity studies are underway.
While PPARgamma inhibitors reduce tubulin levels, older anti-microtubule drug classes; Vinca alkaloids, taxanes (including Taxol) and epithiolones; interfere with microtubule dynamics. To play their role in tumor growth, microtubules must remain flexible. Taxol, for example, "freezes" tubulin subunits, making the cell skeleton rigid and unable to make the shape changes necessary for cell division. Brought to the market by Bristol-Myers Squibb in 1993, Taxol had annual sales of $1.6 billion at its peak in 2000.
Unfortunately, Taxol and other standard, anti-tubulin drugs failed in colorectal cancer clinical trials. Gut tumor cells have evolved to include "efflux" proteins that recognize standard chemotherapies as foreign, and "pump them out" of tumor cells. Even for breast and lung cancer, the tumor types for which Taxol is most used, nearly 100 percent of these tumors eventually become resistant, said Alok A. Khorana, M.D., assistant professor of Medicine at the Medical Center. In cells with more drug efflux pumps, Taxol is not effective (e.g. pancreas, liver and colon), he said.
High-dose PPARgamma inhibitors may overcome the limits of current treatment because the profile of molecules likely to be pumped out by drug efflux proteins is known, and at least one of the PPARgamma inhibitors does not match it. In addition, PPARgamma inhibitors do not bind to the standard tubulin-binding site that renders Taxol useless when binding s ites change shapes thanks to ongoing genetic mutations.
With standard anti-microtubule not an option for colorectal cancer, current chemotherapy regimens feature 5-fluorouracil (5-FU) in combination with other chemotherapy drugs (oxaliplatin, irinotecan) and antibodies (bevacizumab, cetuximab). Once again, response rates of current drugs are low (less than 30 percent as single agents). Tumors generally begin growing again with a year.
Moving forward, the research team will seek to determine exactly which proteins are involved in the anti-cancer effect of PPARgamma inhibitors. Combination therapy is the current leading strategy in treating cancer, and finding the mechanisms by which PPARgamma inhibitors work could be a first step toward their safe combination with other treatments.
"The last work attempting to reduce tubulin levels was abandoned approximately 25 years ago under the assumption that such drugs would be toxic, destroying microtubules in healthy cells as well as cancer cells," Schaefer said. "Our early studies, however, suggest that general toxicity does not rule out this approach as once feared. With new drug delivery technologies that help drugs target only cancer cells, we are very excited about the potential of this line of work."